Diagnostic agent and methods for identifying HIV infected individuals and monitoring their therapy

ABSTRACT

Disclosed herein are methods and a diagnostic agent for identifying HIV-infected individuals. The diagnostic agent, termed C-8.2, whose concentration is altered within about 1-3 days of HIV infection is used in assays not dependent on HIV antigens or antibodies. C-8.2 is present in the serum of all mammals and is a phospholipid or a mixture of related phospholipids.

FIELD OF THE INVENTION

This application is a continuation of U.S. patent application Ser. No.08/065,062 filed May 20, 1993, now U.S. Pat. No. 5,464,740, which is acontinuation-in-part of U.S. patent application Ser. No. 07/940,927filed Sep. 8, 1992 (now abandoned).

This invention pertains to a novel method and diagnostic agent foridentifying HIV-infected individuals. The method can also be used as asurrogate end point to monitor the efficacy of anti-HIV therapeuticagents.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus (HIV, also known as HTLV III andLymphadenopathy virus or LAV) continues to be spread in epidemicproportions throughout the world. HIV is believed to be the causativeagent of Acquired Immunodeficiency Syndrome (AIDS) and AIDS-RelatedComplex (ARC), a prodrome of AIDS. Although the AIDS epidemic may haveleveled off in the United States and the Western World, it continues toincrease in third world countries, especially Africa.

At the present time, the identification of individuals infected by HIVis based on the detection of antibodies and antigens specific for HIV inbody fluids. Antibodies normally do not develop for at least a weekafter exposure to the virus and may not develop for up to 6 months or,in rare cases, two years. Thus, there is a long period of time duringwhich it is not possible to determine if infection by HIV has or has notoccurred. Furthermore, there are many circumstances when the detectionof HIV-specific antibodies cannot be used to evaluate whether anindividual is infected by HIV.

Present screening tests for identifying HIV-infected individuals arebased on the detection of HIV-specific antibodies using variousligand-based techniques, such as an enzyme linked immunosorbent assay(ELISA) as originally developed by Gallo and co-workers in 1984 (Gallo,R. C. et al., Science 224: 500-502, 1984). Because the diagnosis of HIVinfection is so devastating, after an initial positive screening test,HIV infection is confirmed by Western Blot analysis for HIV-specificantibodies. A new method for this confirmation is the demonstration ofthe presence of the viral genome by Polymerase Chain Reaction (PCR)analysis (DeRossi, A. et al., Lancet 2: 278, 1988). This confirmatory,follow-up test is 10 times more expensive than either original test. Inaddition, PCR analysis requires more expertise in its performance, moresophisticated materials and is subject to artifacts if not carefullycontrolled. Therefore, PCR will probably seldom, if ever, be used as aninitial screening test for HIV infection.

Furthermore, there are circumstances when the detection of HIV-specificantibodies is not informative and there is no prior scientific basis fora screening test presently available. Non-limiting examples ofcircumstances where this situation prevails or may prevail are set forthbelow.

For example, babies of HIV-infected mothers may have maternal-derivedHIV-specific antibodies but only 30% to 50% are truly HIV-infected andprogress to develop AIDS (Blanche, S. et al., N. Eng. J. Med.320:1643-1648, 1989). As with adults, PCR and/or tests for HIV-specificantibodies are used in an attempt to discriminate children withmaternal-derived antibodies from infected babies. A positive result byeither technique is perceived to be definitive of infection, but anegative result is not definitive (Borkowsky, W. et al., Lancet 1:1169-1171, 1987). Repeat testing is usually necessary until: (a)maternal antibodies disappear; (b) clinical symptoms develop; (c) T-cellmarker abnormalities are documented; or (d) the newborn reaches 2 yearsof age. Furthermore, both of these techniques are relatively expensiveand, for reasons unknown at the present time, PCR does not alwaysidentify the presence of the HIV viral genome in the first few weeks oflife. In summary, at the present time there is a gap in our ability todetermine which infants born to HIV-infected mothers are trulyHIV-infected and which only have maternal-derived HIV-specificantibodies.

There are at least 6 situations when the determination of HIV-specificantibodies is not informative. In each of these circumstances, a newdiagnostic method is needed.

1. As illustrated above, to screen newborn infants of HIV-infectedmothers.

2. Immediately after the occurrence of high risk events, such as anaccidental needle puncture from an HIV-contaminated sample, prior to thedevelopment of HIV-specific antibodies.

3. As an adjunct for the screening of whole blood for transfusion ormanufacture of blood products. Although all donated blood is currentlyscreened before it is transfused, the screening procedures used are forHIV-specific antibodies employing ELISA and Western Blot analyses. Ifthe donor has not yet developed antibodies (i.e. seroconverted) and isHIV-infected, the disease can be transmitted (Ward, J. W. et at., NewEngland Journal of Medicine 318: 473-478, 1988). In addition, recentlyrecipients of organ donation have been found to be HIV infected. Thisoccurred because the organ donor was HIV-infected and had not yetdeveloped HIV-specific antibodies and was not known to be a member ofone of the high risk groups (male homosexuals, intravenous drug usersand hemophiliacs) for HIV infection.

4. To identify HIV-infected patients after immunization. If an HIVvaccine is approved and used, it will not be possible to distinguishthose individuals who have been vaccinated from those who are trulyHIV-infected. Alternate methods of screening the blood supply will alsobe needed.

5. To identify new therapeutic agents and to monitor their efficacy intherapy as a surrogate end point. Currently, all such monitoring must bedone by analyzing a patient's immune functions (see below).

6. As part of research studies to identify HIV-infected individuals ofother species. For example, vaccine testing is now starting in animalmodels. After vaccination, the animals are exposed to HIV and theinfection is monitored over time. However, because all infected animalshave antibodies induced by the vaccine and may develop other antibodiesfrom HIV exposure, HIV-specific antibody-based techniques cannot be usedto monitor the efficacy of the vaccine. Similarly, new therapeuticagents will be tested in animal models and, again, efficacy cannot bedetermined by simple ELISA, or enzyme immunoassay (EIA) or other testsfor HIV-specific antibodies and antigens because all of the animals willbe infected and have such antibodies and antigens.

Progression to AIDS in individuals with HIV infection is presentlyevaluated by monitoring lymphocyte T-cell markers. Lymphocytes with T-4antigens (also known as CD4) are present in normal (uninfected)individuals in higher concentrations than corresponding cells with T-8antigens (also known as CD8). At the time of infection, and for part ofthe asymptomatic period, T-cell markers are usually within normallimits. Only as HIV infection progresses to AIDS does this ratioreverse; when the number of T-4 cells fall below 400 per ml, mostpatients have clinical features of AIDS. A second gap exists in theknowledge of events and markers for progression to AIDS during theasymptomatic stage of HIV infection. Currently, high levels of β-2microglobulin have been associated with poor prognoses (Mossi, A. R. etal., AIDS 3: 55-61, 1989) whereas elevated levels of urinary and serumneopterin (a folic acid metabolite) have been found in individuals withHIV infection and the levels increase as AIDS develops (Fuchs, D. etal., Immunology Today 9: 150-155, 1988). Thus, although the completecausal relationship has not been fully elucidated, measurement of eithercomponent has been proposed to serve as a prognostic marker for AIDS,but neither can be used for the early detection of HIV infection becausethe above-mentioned changes in concentration occur only a significanttime after infection.

What is needed in the art are new methods for determining whether apatient is infected by HIV in the situations mentioned above in whichthe analysis of HIV-specific antibodies is not informative.

Therefore, it is an object of the present invention to devise methodsfor identifying individuals who are HIV-infected.

It is a further object of the present invention to devise methods toidentify HIV-infected individuals in situations where the detection ofHIV-specific antibodies is not informative or practical.

A still further object of the present invention is to provide adiagnostic agent which can be employed as a surrogate end point formonitoring the therapy of HIV-infected individuals.

SUMMARY OF THE INVENTION

The present invention fulfills the above objects in the discovery of anovel factor, present in the serum of mammals, whose concentration isinitially altered about 1-3 days after infection by HIV. This is a mostimportant finding in that there are FDA-approved treatments forHIV-infected individuals, such as azidothymidine (AZT or Zidovudine) ordideoxyinosine (DDI) which may improve an HIV-infected patient'simmunological functions and substantially delay the onset ofAIDS-related illness in such individuals. Therefore, the earlier that apositive diagnosis of HIV infection can be determined, the sooner suchtherapy can be instituted (Graham, N. M. H. et al., New England Journalof Medicine 326: 1037-1042, 1990).

In one aspect the present invention provides a method for identifying apatient infected with HIV comprising the steps of quantifying the amountof C-8.2 in a blood or a blood fraction sample from said patient;wherein said patient is HIV infected if the amount of C-8.2 in saidpatient's blood or blood fraction sample is statistically different fromthe amount of C-8.2 in blood or blood fraction samples obtained from acontrol, non-HIV infected group.

In another aspect the present invention provides an isolatedphospholipid having a retention time on an amino carbohydrate HPLCcolumn of about 8.2 minutes using an acetonitrile gradient andabsorbance of 210 nm of light.

A further aspect of the present invention provides a method foridentifying a patient infected with HIV comprising the steps ofproviding a blood or blood fraction sample from said patient;quantifying the amount of C-8.2 in said sample; and comparing the amountof C-8.2 in said sample with the amount of C-8.2 present in a blood orblood fraction sample obtained from an age-matched control group ofnon-HIV infected individuals; wherein said patient is HIV infected ifthe amount of C-8.2 in said patient's sample is statistically differentfrom the amount of C-8.2 in said control group.

A still further aspect of the present invention is directed to a methodfor determining the efficacy of the treatment of a human patientinfected with HIV comprising the steps of providing a blood or bloodfraction sample from said patient before said treatment; quantifying theamount of C-8.2 in said sample of step a.; obtaining a blood or bloodfraction sample from said patient during said treatment; or obtaining ablood or blood fraction sample from said patient after said treatment,and quantifying the amount of C-8.2 in said sample of step c.; comparingthe amount of C-8.2 in said sample of step a. with the amount of C-8.2in said sample of step c; wherein said treatment is effective if theamount of C-8.2 in said sample of step a. is statistically differentfrom the amount of C-8.2 in said sample of step c.).

In yet another aspect the present invention provides a method foridentifying a patient infected with HIV comprising the steps ofproviding a serum sample obtained from said patient; quantifying theamount of C-8.2 in said serum sample; comparing the amount of C-8.2 insaid serum sample with that of the amount of C-8.2 present in a serumsample obtained from age-matched control, non-HIV-infected group;wherein said patient is HIV infected if the amount of C-8.2 in saidpatient's serum sample is statistically different from the amount ofC-8.2 in said control, non-HIV infected group.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the present description,claims and drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a High Performance Liquid Chromatography (HPLC) analysis ofserum obtained from a healthy individual.

FIG. 2 is an HPLC analysis of serum obtained from an HIV-infectedpatient with low C-8.2 levels.

FIG. 3 is an HPLC analysis of serum obtained from an HIV-infectedpatient with high C-8.2 levels.

FIG. 4 is an HPLC analysis of C-8.2 levels obtained from a non-HIVinfected newborn infant born to an HIV-infected mother.

FIG. 5 is an HPLC analysis of C-8.2 levels obtained from an HIV-infectednewborn infant born to an HIV-infected mother.

FIG. 6 is an ultraviolet (UV) spectrographic analysis of C-8.2

FIG. 7 (a and b) are Fourier Transform Infrared (FTIR) analyses offraction number 25 isolated from an amino carbohydrate column inaccordance with the methods of the present invention.

FIG. 8 is an FTIR analysis of lipid standards.

FIG. 9 is a Fast Atom Bombardment Mass Spectroscopy (FAB-MS) analysis offraction 24 obtained from an amino carbohydrate HPLC column inaccordance with the methods of present invention.

FIG. 10 is an FAB-MS analysis of sphingosylphosphoryl choline.

FIG. 11 is an FAB-MS analysis of fraction 25 obtained from an aminocarbohydrate column of a sample from a normal individual in accordancewith the methods of the present invention.

FIG. 12 is an FAB-MS analysis of phosphatidyl choline (lecithin).

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited in thisspecification are hereby incorporated by reference in their entirety. Inthe case of inconsistencies, the present disclosure, includingdefinitions, will prevail.

A novel compound, designated C-8.2, which can be isolated, detected,and/or quantitated (by non-limiting example) by HPLC, has been found toundergo acute changes in concentration shortly after HIV infection andcan be employed in a method for identifying HIV-infected individuals.

"Shortly after HIV-infection" is defined herein as about 1-3 days afterHIV infection.

C-8.2 has been found to be present in the serum of humans, pigs, cattle,rats, and mice and is believed to be a component of all mammalian serum.

"Blood fraction" is defined herein as serum or plasma.

Determination of either serum neopterin or β-2 microglobulinconcentration has been proposed for the same purposes as the presentinvention. However, the methods described herein below differ from theexperiments mentioned above in several ways: (1) the ultravioletspectrum of C-8.2 does not contain the characteristic absorption of thepteridine ring characteristic of neopterin; (2) the infrared spectrum ofC-8.2 is not consistent with the presence of a pteridine ring (3) theneopterin concentration does not change within days of exposure to HIVas does the C-8.2 concentration, and (4) β-2 microglobulin is a proteinand has different solubility and physical properties when compared toC-8.2. In contrast, C-8.2 has been identified as a phospholipid or amixture of related phospholipids by isolation and comparison withauthentic materials by Fourier Transform Infrared (FTIR) spectroscopy,ultraviolet (UV) spectroscopy, Fast Atom Bombardment-Mass Spectroscopy(FAB-MS), and HPLC retention time as described below. Thus, C-8.2 isdifferent both from β-2 microglobulin, which is a protein, and fromneopterin, which is a pteridine metabolite. Because neither the β-2microglobulin nor neopterin concentration responds rapidly to theinitiation of HIV infection, neither measurement can be used foridentifying HIV-infected individuals in like manner to C-8.2measurement. Thus, the method disclosed herein for measuring C-8.2levels as a diagnostic indicator for HIV infection could not have beenanticipated by a knowledge of the current state of the art.

Throughout the specification, the marker compound of the presentinvention has been designated as "C-8.2". The present inventor has nowunexpectedly discovered that C-8.2 is sphingomyelin and its derivatives.Derivatives of sphingomyelin include without limitation ceramides(sphingomyelin without the phosphocholine group) andsphingosyl-phospho-choline (sphingomyelin without the acyl amide group).Both C-8.2 and sphingomyelin will be used interchangeably throughout thespecification.

In order to determine the HIV-infection status of a mammal, theconcentration of C-8.2 is determined from blood, plasma or preferablyserum. Determination of the levels of C-8.2 can be accomplished by HPLC,by ligand-based technologies, or by other specific methods known tothose of ordinary skill in the art. In each case, the actualconcentration determined is compared to the normal range for the age (orspecies) of the individual. Statistically significant values eitherabove or below the normal range are diagnostic of HIV-infectedindividuals.

The present inventor has found that the amount of detectable C-8.2varies according to the age of the individual. When practicing themethods of the present invention, the amounts of C-8.2 present in asample are compared to an age-matched control group. The controls can bebroadly grouped as follows: 0 (newborn)-23 months of age; 2 years ofage-17 years of age; and 18 and over.

The uniqueness of the invention is not the particular method fordetermining C-8.2 concentration but the recognition that C-8.2concentration is altered early in the course of infection with HIV andthus that the determination of C-8.2 levels, when compared to normallevels obtained by any technique, can be used to recognize individualswith HIV infection.

It should be understood that when analyzing serum from non-humans, bothHIV and comparable viruses to HIV specific for that species will causethe same perturbations in C-8.2 concentration as has been found inhumans infected with HIV. For example, Simian Immunodeficiency Virus(SIV) infection of primates (e.g. monkeys) can be detected using thesame methods described below for HIV in humans.

C-8.2 refers to the peak detected by High Performance LiquidChromatography (HPLC) using an acetonitrile gradient as the eluant at anelution time of 8.2 minutes after injection of the extracted serumsample into the column. It is the major peak present in thechromatogram. C-8.2 has the following physical properties: it is aphospholipid or mixture of related phospholipids, has a retention timeof about 8.2 minutes when chromatographed on an amino carbohydratecolumn using an acetonitrile gradient and absorbs light at 210 nm andshorter wavelengths. As shown below in Example 4, C-8.2 has beenidentified as sphingomyelin and its derivatives. These physicalproperties confirm its identity.

Using the method of the present invention, a positive diagnosis of HIVinfection is made when the levels of C-8.2 are substantially higher orlower (also referred to herein as statistically different) than thosedetermined in age (and species)-matched control groups. "Substantiallyhigher or lower" or "statistically different" levels of C-8.2 aredefined herein as three standard deviations above or below the mean ofthose determined in healthy (non-HIV-infected) controls. Such standarddeviations can be obtained using statistical methods well-known to thoseof ordinary skill in the art. The controls do not necessarily have to beperformed simultaneously with every assay. A normal (control) range canbe determined once for each patient population or group (as disclosedabove) and the results of each assay compared to the values determined.

Using the method of the present invention a blood fraction, serum, forexample, is obtained from a patient suspected of being infected by HIV.The minimum amount of serum needed for this analysis is 0.10 ml. Theserum is extracted with an organic solvent, such as methanol, ethanol,tetrahydrofuran, chloroform or preferably acetonitrile at a ratio ofbetween about 2 and 5 to 1 (organic solvent: serum) and preferably 4:1.The extract is centrifuged to resolve an organic and aqueous phase, forexample 750×g for about 5 minutes at between about 2° C. and about 4° C.The organic layer is saved and contains C-8.2.

The amount of C-8.2 is then determined from the above mentioned organicphase, by non-limiting example, by HPLC analysis or preferably,antibody-based assays such as those described in Example 2 below.Antibody-based assays are preferred due to their ease of performance andthe fact that sophisticated equipment is not required.

Many methods for the utilization of a specific antibody and tracer forthe quantitation of a ligand have been developed. For radioimmunoassay,dextran-coated charcoal may be used to absorb unbound tracer, thuspermitting determination of the bound fraction. Alternatively, (a) thebound fraction may be collected by binding to a second antibody(specific for IgG), either in solution or chemically bound to beads; (b)polyethylene glycol or ammonium sulfate may be added to enhanceseparation of bound and free tracer; or (c) coated tube methodologycould be applied to permit separation of bound and free tracer. Inaddition to radioimmunoassay, the amount of phospholipid may bedetermined by Immunoradiometric assay (IRMA) (Miles, L. E. M., et al.Lancet. 2: 492, 1968), enzyme immunoassay (EIA) (Berry, N. J. et al., J.Virol. Met. 34: 91-100, 1991), and ELISA (Engvall, E. et al., G.Immunochemistry 8: 871, 1971), and any other ligand assay techniqueknown to those of ordinary skill in the art.

Suitable HPLC columns for use in the present invention includeAdsorbosphere - NH₂ (Alltech, Chicago, Ill.) Econosil - NH₂ (Alltech),devitalized polydextran beads, such as Lichrosorb - NH₂ (Alltech),SEPHADEX™-LH20 (Pharmacia, Piscataway, N.J.) and preferably an AminoCarbohydrate column (Alltech). The chromatographs are compared with age(and species, where applicable)-matched controls. Any value 3 standarddeviations above or below the control values is indicative of a positivediagnosis of HIV infection.

The solvent to be used with each of the above HPLC columns can beoptimized for C-8.2 detection and quantitation. For example, whenemploying an Amino Carbohydrate column, a complex gradient can be usedsuch as t=0 minutes (time post injection of the sample into the column),95% acetonitrile; t=2 minutes, 75% acetonitrile; t=10 minutes, 55%acetonitrile; t=12 minutes, 40% acetonitrile; t=14 minutes and t=15minutes, 95% acetonitrile (elution). When employing the other HPLCcolumns disclosed above or any other equivalent affinity column, suchbinding, washing and elution gradients can be determined by routineexperimentation well-known to those of ordinary skill in the art basedon the physical properties of C-8.2 disclosed herein.

As described below, the C-8.2 levels determined in HIV-infectedindividuals were found to return to normal upon the initiation of AZTddI or ddC therapy only when such therapy was effective. As describedbelow four HIV-infected children, ages 4-11 years, had neurologicaldysfunctions typical of HIV infection. All of the infected children hadstatistically significantly elevated levels of C-8.2 in their serum asdetermined by HPLC analysis. All the children were given AZT in order tocontrol disease symptoms. AZT therapy was ineffective in all fourchildren and the levels of C-8.2 didn't change. However, in eightage-matched children who were also HIV-infected and received AZT andbenefitted from the treatment, C-8.2 levels returned to the normalrange. Therefore, determination of C-8.2 levels can be used to monitorthe efficacy of anti-HIV therapeutic agents.

When used to monitor efficacy of antiretroviral drug treatment using,for example, AZT, ddI, ddC or mixtures thereof, C-8.2 levels can bemonitored during or after the institution of such therapy. These levelscan be compared to the levels determined before therapy has started.Therefore, if the C-8.2 levels do not return to those obtained forage-matched control groups, therapy may be discontinued. This is a mostimportant finding because all anti-HIV theraputics are highly toxic andif they are not effective they should be discontinued as soon aspossible.

The method of preparing serum extracts for analysis is suitable forblood or plasma. Larger or smaller volumes can be used as long as therelative proportions disclosed above are maintained. Because of thespecific hazard of HIV transmission, it should be presumed that allhuman materials are from HIV-infected patients and the handling of bloodor tissue specimens should be performed with great care.

In addition to its utility as a marker of HIV infection, theidentification of the abnormal regulation of C-8.2 levels and itstemporal relation to infection by the virus suggests that it may be usedas a surrogate end point or measurement of the health or infectionstatus of a patient to evaluate therapy.

Without wishing to be bound by theory, it is believed that for thelevels of C-8.2 to change so rapidly as one of the early events afterHIV infection, there should be a C-8.2-specific hydrolytic or syntheticenzyme whose activity is altered upon HIV infection. The postulatedenzyme could be part of the process whereby the cell machinery issubverted by the virus. Blocking of the process may prevent the virusfrom controlling cellular functions. As such, the recognition of theexistence of such an enzyme can be used to identify a possible newtarget for HIV-specific chemotherapy.

The assays of the present invention are based on alterations in thelevels of C-8.2 and not on antigenic properties of the virus. Therefore,infection by all Human Immunodeficiency Viruses (HIV-1, HIV-2, etc.)will be detectable using these assays, as will any other antigenicvariants thereof. Indeed, one of the advantages of the method fordetecting HIV infection of the present invention is its ability todetect all HIV irrespective of their antigenic makeup. This is adistinct advantage because HIV has been found to be highly mutagenic. Asthe virus changes, no new reagents will be required to practice themethods of the present invention.

HIV is not stable in organic solvents. The solvent extraction methoddescribed above (a) inactivates free HIV particles and (b) destroysHIV-infected cells. These are the agents by which HIV is transmitted.Thus, the use of this step reduces the hazard to the technologistperforming the assay.

C-8.2 is believed to be a phospholipid or mixture of relatedphospholipids based upon the FTIR shown in FIG. 8 (a and b) because thespecific bands in the lipid standards shown in FIG. 9 are also presentin C-8.2.

The first example below will describe the use of HPLC for the rapiddetermination of C-8.2 concentrations. The second example will describethe production of ligand-specific materials and methods for C-8.2detection. The third example will describe the preliminarycharacterization of C-8.2 obtained from normal adults.

The present invention is further described below in specific workingexamples which are intended to further illustrate the invention withoutlimiting its scope.

EXAMPLE 1 Determination of C-8.2 Levels by HPLC

Serum (0.1 ml) was extracted with acetonitrile (0.4 ml) in a glass tube(12×75 mm) at room temperature. The extract was centrifuged at 2°-4° C.for 5 minutes at 750×g. The extract was removed with a 1 ml syringe, thesyringe attached to a filter (0.45 microns, LC13 Acrodisk, GelmanLaboratories, Ann Arbor, Mich.) and the extract filtered. The filtratewas collected in a suitable glass vial. If desired the vial may be anHPLC autosampler vial that matches the HPLC autosampler if one is used.

HPLC Analysis:

The method for HPLC (Model 401T, BioRad, Richmond, Calif.) analysis isbased on the use of an amino carbohydrate column (4.6×250 mm; Alltech,Chicago, Ill.). Samples were eluted with a complex gradient usingmixtures of acetonitrile and water: (1) t=0 minutes, 95% acetonitrile;(2) t=2 minutes, 75% acetonitrile; (3) t=10 minutes, 55% acetonitrile;(4) t=12 minutes, 40% acetonitrile; (5) t=14 minutes, 40% acetonitrile;(6) t=15 minutes, 95% acetonitrile. Eluted materials from thechromatogram were detected by monitoring ultraviolet absorbance at 210nm. Peak areas and heights were determined with a computer softwarepackage (BioRad). Both height and area (in area integration units--AU)were proportional to the amount of extract injected. Normal values maybe determined with either parameter.

Results: Adults

Serum was obtained from normal (non-HIV-infected) adults, extracted andchromatographed as described above. Serum concentrations of C-8.2 innormal adults of both sexes (n=14) had an approximately normaldistribution with a mean of 38 area units (AU) and a standard deviationof 3.5 AU. Samples were scored as normal if they were within the rangeof 27 to 48 AU and abnormal if they were not within three standarddeviations, 10.5 AU, within this range (a representative chromatogram isshown in FIG. 1). Based on a normal distribution, 99% of samples shouldbe within this range. Abnormal samples can be lower or higher than thenormal range.

C-8.2 in serum from adults with a known HIV infection (n=19), as shownby presence of HIV-specific antibodies, ranged from 5 to 65 AU with amean of 38 AU; there were few samples within the normal range (n=3) andthe values were not normally distributed (representative chromatogramsare shown in FIG. 2 and FIG. 3) The box below shows the correlation ofHIV status as determined by specific enzyme immunoassay, with scores forC-8.2 within 3 standard deviations the normal range and not within threestandard deviations the normal range.

First Group:

    ______________________________________                                        HIV antibodies detected by EIA                                                HIV by HPLC       NEGc    POS.sub.d                                           ______________________________________                                        NEG.sup.a         14      3                                                   POS.sup.b         0       16                                                  .sup.1 X.sup.2  = 22.9; p < 0.0001                                            ______________________________________                                         .sup.a C-8.2 concentration determined by HPLC within the range 27 to 48       .sup.b C8.2 concentration not within range 27 to 48 AU                        cHIV negative serum when tested by EIA                                        .sub.d HIV positive serum when tested by EIA                                  .sup.1 Chi Square.                                                       

Therefore, the above results show that the method of the presentinvention was able to identify 16 out of 19 HIV-infected individuals. Itremains to be seen if the 3 antibody positive patients with normal C-8.2levels actually develop AIDS at a later date.

Second Group:

The samples tested (n=37) were selected from samples obtained from adultindividuals at a high risk for HIV infection (male homosexuals,intravenous drug abusers, or their sexual partners) and tested for thepresence of HIV antibodies by a commercial blood bank. The C-8.2 levelsranged between 28 AU and 48 AU with a standard deviation of 3.5 AU.

    ______________________________________                                        HIV antibodies detected by EIA                                                HIV by HPLC       NEG     POS                                                 ______________________________________                                        NEG               13      4                                                   POS               7       13                                                  X.sup.2 = 6.36; p < 0.01                                                      ______________________________________                                    

The above illustrates the general utility of the method of the presentinvention for identifying individuals infected with HIV.

Third Group:

Most serum samples (8 of 10) obtained from adults with HTLV 1 or 2infection (n=10) were within the normal range of the samples². Of the 2samples that had C-8.2 levels out of the normal range, one testedpositive for HIV by EIA; the other did not, but there was no furtherfollow-up possible to determine if the patient was in the time periodafter infection but prior to development of antibodies. The dataillustrates that the change in C-8.2 concentration is specific for HIVrather than for other types of related human T-cell lymphotrophicviruses.

Fourth Group:

Two sero-conversion panels with twice weekly samples were tested asshown in the box below. The samples were obtained from two adults whohad unidentified "high risk" events and agreed to provide serum samplestwice a week. In each case the first sample was obtained within 48 hoursof the event (no other information about the nature or timing of theevent was made available in order to preserve confidentiality). NormalC-8.2 levels for these patients should be between 28 AU and 48 AU with astandard deviation of 3.5 AU. The results are set forth in Table 1below.

                  TABLE 1                                                         ______________________________________                                                             HIV-specific                                                        C-8.2 Level Antibodies                                                        Patient                                                                              Patient  Patient  Patient                                   Sample #                                                                              Day #    1        2      1      2                                     ______________________________________                                        1       1        25.5     25.1   0      0                                     2       4        19.8     15.0   0      0                                     3       7        23.5     23.4   +      0                                     4       10       24.5     --     ++     0                                     5       14       26.2     16.1   +++    0                                     6       17       30.0     15.0   +++    0                                     7       21       24.3     14.8   +++    0                                     8       24       25.2     --     +++    0                                     ______________________________________                                    

In both individuals, the initial concentration of C-8.2 (on day 1) wasalready three standard deviations below the normal range and thereforediagnostic of HIV infection. In the first patient, when HIV-specificantibodies developed, the C-8.2 level returned towards normal but thatvalue was not sustained as the infection progressed. In the secondpatient, HIV-specific antibodies did not develop for several months andno rebound in C-8.2 level was observed. In both patients, the change inC-8.2 level occurred prior to the development of HIV-specificantibodies.

These data illustrate the utility of C-8.2 level determination forrecognizing HIV-infected individuals within 3 days of a high risk eventthat might lead to HIV transmission.

Results: Infants With HIV Antibodies

Group 1: Normal Infants

C-8.2 levels in normal (non-HIV-infected) infants (ages 8 days to 15months) ranged from 18 to 33 AU (n=11) with a mean of 25.5 and astandard deviation of 2.5 AU. This sets the normal range for comparisonwith the infants at high risk for HIV infection. A representativechromatogram is shown in FIG. 4.

Group 2: Infants Less Than 18 Months Of Age At High Risk For HIVInfection

In serum from high-risk infants (born to HIV-infected mothers) less than18 months of age (n=27) with HIV-specific antibodies at birth, levels ofC-8.2 were within the normal range in 15 and were out of the normalrange in 12. None of the children with C-8.2 values within the normalrange had symptoms of HIV infection, while 9 of 12 of the infants out ofthe normal range had clinical signs of HIV infection, includinglymphadenopathy. The final diagnosis of the remaining 3 is not yet knownbecause of their age, although they continue to have HIV specificantibodies in their serum. A typical chromatogram is shown in FIG. 5.The data are tabulated below.

    ______________________________________                                        Infants less than 18 months of age                                            at high risk for HIV infection                                                C-8.2 Level Determined by HPLC                                                              No symptoms                                                                              Symptoms                                             ______________________________________                                        Normal.sup.a  15         0                                                    Abnormal.sup.b                                                                              3          9                                                    X.sup.2 = 16.9; P < 0.001                                                     ______________________________________                                         Notes:                                                                        .sup.a C8.2 concentration within the range 18 to 33 AU                        .sup.b C8.2 concentration either three standard deviations below or above     33 AU                                                                    

Group 3: Older Infants And Children

In older infants and children, aged 2 years to 11 years, C-8.2concentrations were statistically significantly elevated (i.e. at least3 standard deviations) in all (n=4) HIV-infected subjects untreated withAZT (results not shown). In AZT-treated individuals (n=16), C-8.2 waswithin the normal range in 5 subjects and elevated in 11 subjects(results not shown). The latter group continued to have neurological andbehavioral disorders and other neurological symptoms typical of HIVinfections, whereas the group with normal values did not have suchdisorders. Importantly, in 2 children, sequential samples showed a highC-8.2 level prior to AZT therapy which decreased to the normal rangewithin a month of the start of treatment.

In summary, C-8.2 determination in infants with HIV-specific antibodiesdiscriminated between those infants with antibodies of maternal originfrom those infected with HIV. Secondly, C-8.2 levels were a marker forthe effectiveness of AZT therapy.

Evaluating C-8.2 Levels for Monitoring the Efficacy of AntiretroviralDrug Therapy

Four HIV-infected children, ages 4-11 years had neurological symptomstypical of HIV infection. Their serum levels of C-8.2 were significantlyelevated (40, 41, 44 and 46 AU) when compared to age-matched controls(20-33 AU, N=11). AZT therapy was instituted but was not effective inreducing the disease symptoms and C-8.2 levels didn't change in thesepatients. However in 8 HIV-infected children with C-8.2 levelssignificantly different from control values receiving the same AZTtreatment who benefitted from AZT, C-8.2 levels returned to normalranges (24-34 AU).

In addition, a four year old child who was HIV antibody negative, astested by ELISA and Western Blot, was examined. The C-8.2 levels were 41AU. The child was symptomatic for HIV infection and could be diagnosedas HIV positive using the methods of the present invention.

EXAMPLE 2 Development Of Reagents For Ligand Assays Method ForGeneration Of C-8.2 As A Hapten Linked To Carrier Protein

Described below are methods for the preparation of reagents for ligandbased assays for quantitating C-8.2.

The fatty acid side-chain of C-8.2 contains several alkene groups. Thesealkene groups add iodine or other halogens across the alkene bond. Theresulting iodine can be displaced with mercaptoacetic acid (Aldrich,Milwaukee, Wis., cat. number M310-8) or 3-mercaptopropionic acid(Aldrich, cat. number M580-11). The difference between the two reagentswill be the length of the spacer chain between the carrier and thehapten. Longer spacer chains may also be used. The products may bepurified by chromatography on a DEAE derivative of a polydextran beadsuch as SEPHADEX™ A-50 (Pharmacia, Piscataway, N.J.) withacetonitrile/water (1:1) as the eluant, derivatized polydextran beadssuch as SEPHADEX™ LH-20 (Pharmacia) with methanol as the eluant, andfinally with SEPHADEX™ a polydextran molecular sieve column such as G-10(Pharmacia) with water as the eluant. The C-8.2 used as the startingmaterial may be isolated from normal plasma. The thioglycolatederivative of C-8.2 may be coupled to Bovine Serum Albumin (BSA) withEDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride).Alternately, a complete kit for coupling the resulting product tothioglycolate can be used (Pierce, Rockford, Ill.; cat #77101 A).

The above conjugates can be used to raise specific antibodies directedagainst C-8.2. These antibodies can be either polyclonal (Lieberman, S.et al., Recent Progress in Hormone Research 15: 165-200, 1959) ormonoclonal (Blethen, S. L. et al., J. Ped. Endocrin 3: 217-223 (1989).

Methods For Preparation Of A Tracer

A method for the labeling of phospholipids has been published (AnthonovP. A. Panmcheva R. P., Ivanov I. G. Biochem. Biophys. Acta 835: 408-410,1985). In this method, 1 mCi of Na [¹²⁵ I] (Dupont, Boston, Mass.) in 10μl of water is mixed with 10 μl of 0.3M sodium acetate, pH 4.5 and 10 μlof freshly prepared 3.0 mM TlCl₃. The volume is adjusted to 100 μl. Themixture is allowed to react for 5 minutes and 10-15 mg of C-8.2 is addedas a solution (0.5 to 1.0 ml) in chloroform/methanol (1:1). The mixtureis allowed to incubate at room temperature for 10 to 15 minutes. Thereaction is quenched by the addition of 3 ml of chloroform and 5 ml of20 mM Na₂ S₂ O₃. The mixture is vortexed and centrifuged at 250×g for 5minutes. The lower, organic layer will contain the lipids. The lipidproduct is washed twice with 5 ml of 20 mM Na₂ S₂ O₃. The chloroformextract is evaporated to dryness and purified by thin layerchromatography (Whatman, Alltech, Chicago, Ill.) or a derivatizedpolydextran beads such as SEPHADEX™ LH-20 column chromatography. Thefinal product will have a specific activity of about 10 μCi per mg ofphospholipid.

Alternatively, the reaction can be catalyzed by Chloramine T (PierceChemicals) as follows. In this method, 1 mCi of Na[¹²⁵ I] (DuPont,Boston, Mass.) in 10 μl of water is mixed with 10 μl of 0.1M phosphatebuffer, pH 7.2. Chloramine T (10 μl of a 20 mM solution) is added andthe volume adjusted to 100 μl. The reaction and purification will becarried out as described above.

An enzymatic method of catalyzing the introduction of iodine intophospholipids has also been reported (Benenson A. Mersel M. Pinson A.,and Heller, M. Anal. Biochem. 101: 507-512, 1980). In this procedure,the specific phospholipid is subjected to ultrasonic radiation inCa-Mg-free Phosphate-Buffered-Saline (PBS, pH=7.4). To the phospholipidis added 100 μl of 0.1 mCi carrier-free [¹²⁵ I] (DuPont, Boston, Mass.),50 μl of lactoperoxidase (0.25-2.5 μg, BioRad, Richmond, Calif.), 50 μlof 300 μU glucose oxidase (BioRad) and 250 moles of glucose in 50 μl.The mixture is incubated at room temperature for 15 min. The reaction isquenched by the addition of 10 μl of 6.6 mM thiosulfate (FisherChemicals, Springfield, N.J.) and the product purified as above.

Methods For Utilization Of Antibody And Tracer

A typical antibody-based assay may be performed as follows:

1. Initial detection and titration of antibody:

Sera from rabbits immunized with C-8.2-BSA conjugates (or monoclonalantibody containing media or fluid) is serially diluted with bovineserum albumin (BSA) in phosphate buffered saline, pH=7.4. The tracer, asprepared above, is diluted with the same buffer to a concentration of40,000 to 125,000 CPM/ml. The serial dilutions are pipeted into 12×75 mmglass tubes and incubated with the tracer (0.4 ml) for 1 hour at 37° C.At the end of the incubation, the tubes are placed in the refrigerator(2° to 4° C.) for 20 minutes. Rabbit IgG specific Immuno-beads areadded. (Bio-Rad, Richmond, Calif.) (If monoclonal antibodies used, thenthe Immuno-beads should be specific for mouse IgG.) Each tube isvortexed and allowed to incubate for 1 hour in the refrigerator at 2°-4°C. At the conclusion of the second incubation, the tubes are centrifugedin a refrigerated centrifuge at 2000×g for 20 minutes and thesupernatant discarded without disturbing the pellet. Each tube is thencounted in a gamma counter (LKB, Gaithersburg, Md.). The initialdilution of antibody to use for assay is the dilution that produceshalf-maximal amount of binding. For the most sera obtained aftersuitable immunization, the active dilution to be used ranges from1/1,000 to 1/50,000, for a final dilution of 10,000 to 500,000 per ml ofserum. After establishment of the standard curve, the final dilution forthe antibody is further modified to control the sensitivity, asdescribed below.

2. Establishment of standard curves

Pure C-8.2 (as isolated in Example 3 below) is diluted with BSAcontaining phosphate buffered saline and used to generate a standardcurve. The actual amount used for each standard is confirmed by the HPLCmethod (Example 1). The range of the standard curve is established basedon the normal values obtained using the method of Example 1. Standard 4has the mean amount of C-8.2 detected in the control population.Standard 2 and 6 have 3 standard deviations less or more, respectively,than the mean amount detected in the control population. Theconcentration in the remaining standards are selected to maintaingeometric proportion. The final antibody dilution is adjusted such thatstandard 4 displaces 50% of maximum binding (Bo) as measured in tubes5,6. The specific activity of the tracer is adjusted (lowered) byaddition of unlabeled material to adjust the slope of the standard curveto satisfy these parameters.

    ______________________________________                                        3. Assay Protocol                                                             Tube # Description    Comments                                                ______________________________________                                        1,2    Non-Specific Binding                                                                         No Antibody                                             3,4    Total          Tracer only, reserve for                                                      counting                                                5,6    Maximum binding (Bo)                                                                         Antibody and Tracer (A + T)                             7,8    Std.   1 (10 μl)                                                    9,10   Std.   2 (10 μl)                                                    11,12  Std.   3 (10 μl)                                                    13,14  Std.   4 (10 μl)                                                    15,16  Std.   5 (10 μl)                                                    17,18  Std.   6 (10 μl)                                                    19,20  Std.   7 (10 μl)                                                    21,22  QC -   1 (10 μl)                                                                              Value established as low end                                                  cut-off                                             23,24  QC -   2 (10 μl)                                                                              Value established as                                                          expected mean                                       25,26  QC -   3 (10 μl)                                                                              Value established as high                                                     end cut-off                                         27,28  Unknown (10 μ1)                                                                           From individuals to be                                                        tested                                                  etc.                  Additional tubes as needed                              ______________________________________                                    

i. Label tubes for the above protocol.

ii. Piper 10 μl of each standard, control and unknown into theappropriate pair of tubes.

iii. Add 400 μl of tracer to each tube with a repeating pipet. Put asidetubes 3 and 4; they are ready for counting.

iv. To tubes 1 and 2, add 100 μl of phosphate buffered saline with BSA,pH=7.4.

v. Starting with tube no. 5 above, add 100 μl of antibody diluted asabove to each tube.

vi. Vortex all tubes. Incubate 1 hour at 37° C.

vii. Transfer all tubes to refrigerator (2° to 4° C.) and incubate for30 minutes.

viii. Dissolve Immunobeads (20 mg) in 20 ml of phosphate bufferedsaline. Mix slowly on stirrer at 2° to 4° C. until needed.

ix. Add 100 μl of Immunobeads to each tube. Vortex. Incubate inrefrigerator for 30 minutes.

x. Centrifuge tubes at 2000×g (3200 RPM) for 20 minutes at 2° to 4° C.(RT6000 B Dupont Centrifuge, Dover, Del.).

xi. Decant liquids without disturbing pellet. Count tubes in gammacounter.

4. Sample Preparation

All blood samples should be treated according to the OSHA blood bornepathogen standard. To prepare samples for assay, whole blood iscentrifuged in a covered centrifuge for 5 minutes at 750×g. The clearserum is transferred to a suitably labeled glass tube, capped and storedfrozen at -70° C. until assayed. At the time of assay, the sample isthawed at room temperature and vortexed carefully. No heat should beused in thawing. Samples must not be allowed to remain unfrozen for longperiods.

5. Analysis of Results

i. The standards are used to generate curves as for other RIA procedures(plot (a) counts bound vs. concentration, (b) bound counts vs. log ofconcentration, or (c) log of bound counts vs. log of concentration).Interpolate unknowns against the standard curve. The calculations can beperformed by hand or computer as desired.

ii. Data sets are accepted as valid and results reported only when theQC samples are within the expected ranges.

iii. Diagnosis of HIV infection should be confirmed by alternatetechniques, such as PCR or Western blot. In the event diagnosis cannotbe confirmed by one of these techniques, additional samples should beobtained and assayed.

In addition to RIA, the concentration of C-8.2 in Serum can bedetermined with other assays based on specific antibodies (such as IRMA,EIA, or ELISA) or by other standard color development techniques, suchas those used in clinical chemistry for the assay of cholesterol.

EXAMPLE 3 Characterization Of C-8.2 From Normal Individuals DescriptionOf Assay For Purification

Quantification of the amount of C-8.2 in a particular fraction wasperformed by injection of an appropriate aliquot onto the HPLC column asdescribed in Example 1 above. The peak area (in area units--AU), asdetermined by computer integration, is then multiplied by the dilutionfactor to determine the total amount of C-8.2 in AU.

Isolation Of C-8.2 From 1.5 Liters Of A Plasma Pool And ChemicalAnalysis

A plasma pool (1.5 L) obtained from normal, non-HIV infected adults, wasextracted with 6 L of acetonitrile. The extract was filtered to removedenatured and insoluble materials. The extract was treated with 3.75 Lof benzene and 2 phases formed: (1) the lower, aqueous phase -discarded-and (2) the upper, organic phase. The upper phase was evaporated todryness, in batches, on a rotary evaporator (Buchler, purchased fromFisher, Springfield, N.J.) with the temperature of the water bath notexceeding 40° C. Each batch was dissolved in a limited amount ofmethanol and pooled with other methanolic extracts. The final pooledmethanolic extract was reduced in volume to less than 10 ml.

The second step was chromatography derivatized polydextran beads such ason SEPHADEX™ LH-20 (Pharmacia, Piscataway, N.J.) with methanol as thesolvent. C-8.2 was purified in 5 ml batches and corresponding fractionspooled. C-8.2 concentration of each fraction was monitored by HPLC. Arepresentative chromatogram is shown in FIG. 4. Fractions containingC-8.2 were pooled and evaporated to dryness in a rated V-vial (Fisher,Springfield, N.J.) under a stream of dry nitrogen. The V-vival wasweighed, partially purified C-8.2 was dissolved in a minimal volume ofmethanol and the amount of product determined by HPLC. The fractionswith the largest peaks of C-8.2 had the smallest peaks of othermaterials. Because the wavelength measured was 210 nm and thecontaminants absorbed light predominantly at other wavelengths, it wasnot possible to determine the actual degree of purification.

The fractions with the highest specific activity were fractions 9-11with some C-8.2 also present in fractions 12-14. These side fractions oflower specific activity were reserved. Only the high specific activityfractions (9-11) were used for further purification.

The final step in purification of C-8.2 was preparative HPLC with an NH₂-Lichrosorb column (10×250 mm, Alltech). Material applied to column in95% acetonitrile - 5% water. C-8.2 eluted at linear gradient to 40%acetonitrile - 60% water. A chromatogram with the same gradient as usedfor serum samples with a single peak at 8.2 minutes was obtained,indicating that the material seemed to be homogeneous by this criterion(data not shown).

Based on the specific activity (AU/mg) of C-8.2 isolated from 1.5 litersof a plasma pool from uninfected adults, the approximate concentrationof C-8.2 was 100 mg/1 or about 125 μM.

UV Analysis

The UV spectrum of C-8.2 were determined with a model lambda 3a UV/VISspectrophotometer (Perkin-Elmer, South Plainfield, N.J.). The sample wasprepared as follows: The material was prepared in 80% acetonitrile - 20%water. The same solution was used as the reference. This solvent mixturedoes not absorb light with wavelengths longer than 200 nm.

The UV absorption spectrum of C-8.2 is shown in FIG. 6. The significantportions of the spectrum are maximum absorption of 210 nm and shorterwavelengths and the absence of peaks in the range of 220 to 280 nm. Theabsence of the peaks at these wavelengths confirms the absence ofaromatic groups such as amino acids, nucleic acids or pteridines.

FTIR Analysis

The Fourier Transform Infrared (FTIR) analysis spectrum of C-8.2 isshown in FIG. 7 (a and b). The spectra were obtained by a commerciallaboratory (Hauser Chemicals, Golden Colorado).

The sample was dissolved in methanol and introduced to the FTIRspectrometer as a KBr pellet. A second spectrum was acquired using themicroscope attachment. The spectra obtained were compared to referencespectra from known libraries for possible identification.

FIG. 7a represents the spectrum acquired on the sample prepared as a KBrpellet. FIG. 7b represents the FTIR spectrum acquired on the same sampleusing the microscope. FIGS. 8a and 8b are substantially identical. FIG.8 represents a series of library spectra which comprise the five bestfits from the available spectra data base.

The FTIR spectrum strongly suggested the presence of a carbonyl from anester. The carbonyl was judged not to be conjugated. Additionally, thestrong absorbance at 1249 cm⁻¹ is a positive indication of the presenceof a P-O functional group. The broad absorbance at 3278 cm⁻¹ may beattributed to a hydroxyl functional group.

FAB-MS Analysis

The mass spectrogram of C-8.2 is shown in FIGS. 9 and 11.

FIG. 9 depicts the FAB-MS spectrum of Fraction 24 mentioned above. InFIG. 9, the peak at 572 is from Gramicidin, added as an internalcontrol. FIG. 10 depicts the FAB-MS spectrum of an authentic standardfor spingosylphosphoryl choline (SPC The inset in FIG. 10 is anenlargement of the area of the control peaks. Comparing FIGS. 9 and 10,it can be seen that Fraction 24 contains SPC (having a peak at 466.7).

FIG. 11 depicts the FAB-MS spectrum of Fraction 25 as is lated fromadult human serum. In FIG. 11, peaks at 523 and 572 were generated frominternal standards added to Fraction 25 before analysis. The spectrum ofFraction 25 also has a peak at about 466, indicating that one componentof C-8.2 is SPC.

FIG. 12 shows the spectrum for phosphatidyl choline (lecithin). Theabsence a peak at 760 in FIG. 11 (Fraction 25) and the absence of a peakat 497 (observed in FIG. 11) confirms that lecithin is not a componentof C-8.2.

From the above FAB-MS analysis, it was concluded that SPC is a componentof C-8.2.

Additional Purification Procedure

In order to get enough material to identify the structure of C-8.2, theisolation method was scaled up to 10 liters of serum.

The initial extraction method (mixing with 4 volumes of acetonitrile)was a duplicate of the method used above. The extract was thenpartitioned by the addition of benzene. The upper, organic, phase wasevaporated to dryness and chromatographed on SEPHADEX198 LH-20(Pharmacia, Piscataway, N.J.) with methanol as the solvent. All of thefractions containing material that eluted at 8.2 mins. (Nos. 8-12) werecombined and concentrated by evaporation under vacuum. The pooledfractions were subjected to preparative HPLC (2 ml total in 0.35 mlbatches). Each fraction was tested by analysis of an aliquot with theanalytical HPLC column method described above. Material was found in thefollowing fractions: (a) 24, (b) 25, (c) in lower concentrations in26-31 designated as P2, and (d) in fractions 50 to 60 designated as P1.Mixing fractions did not indicate the presence of any doublets on theanalytical column.

An aliquot (5% of the total) of fraction 25 was analyzed by FTIR. Thepattern obtained was consistent with a phospholipid,sphingophosphoryl-choline but the complete structure of the lipid andits fatty esters could not be identified.

EXAMPLE 4 Identity of Surrogate Marker For HIV Infection

With the HPLC assay procedure described above, a large amount (100 mg)of C-8.2 was purified to apparent homogeneity from bovine blood (5liters). The molecular weight of the product was determined to be amixture of 758 and 786 daltons. This could be generated by the additionof linolenic acid as the acyl amide of sphingosyl-phospho-choline andthe corresponding compound with 2 additional carbons. These structureshave the trivial name of sphingomyelin. Sphingomyelin is not limited tolinolenic-sphingosyl-phospho-choline, but also other fatty acid amidesof sphingosy-phospho-choline. Authenticpalmitoyl-sphingosyl-phospho-choline was obtained from Sigma ChemicalCo. (St. Louis, Mo.) and used as a model compound. This authenticmaterial had the same HPLC retention time as the isolated material (datanot shown). FTIR was also consistent (data not shown). When analyzed byproton NMR, the model compound and the isolated material (i.e. C-8.2)had closely related spectra (data not shown).

Sphingomyelin is a major phospholipid component of cell membranes. Thestructural elements are: N-acyl-sphingosyl-phosphocholine, where theacyl group is usually either a 16 or 24 carbon fatty acid. In spite ofhigh serum levels (in excess of 400 μmol/L or 35 mg/dl), the enzymes andregulatory mechanisms for both its synthesis and degradation are poorlycharacterized. Sphingomyelin is required for differentiation ofmacrophage/monocyte cells in response to phorbol esters. This process isprotein kinase C dependent and inhibited by sphingosine. Thus,sphingomyelin and its precursors have an intimate role indifferentiation of monocytes and may have a similar role in lymphocytes,including CD4+ cells.

Sphingomyelin is degraded to ceramides by the action ofsphingomyelinase, which is an extracellular plasma membrane enzyme andis present in high concentrations in brain, liver and adrenals.Niemann-Pick syndrome is caused by an inherited defect in any one offive enzymes catalyzing degradation of sphingomyelin to ceramides, thusleading to high levels of sphingomyelin in serum. Individuals with thissyndrome have an increased risk of developing autoimmune disorders,which may be caused by increased lymphocyte proliferation secondary toincreased sphingomyelin levels.

Serum sphingomyelin levels are subject to tight metabolic control asillustrated by (a) the observed tight range in observed concentrations(35±3.5 mg/dL), (b) the lack of day to day variation and (c) the lack ofchange in concentration after a meal. However, the biochemical basis forthis regulation is entirely unknown.

As described here, the observed natural course of sphingomyelin levelssubsequent to HIV infection is (a) an immediate transient decrease, (b)a sustained increase when HIV-specific antibodies develop and (c) areturn towards normal levels when AZT therapy is effective. Theaforementioned increase is a consequence of the return of physiologicalcontrol in the absence of HIV, in turn secondary to the antibodyproduction. Serum sphingomyelin levels then increase until supranormallevels are attained. In response to AZT, sphingomyelin levels returntowards normal levels from either extreme. These changes point to HIV asmodulating the specific control pathway for serum sphingomyelin levels.If this were the case one could predict that there would be changes inlymphocyte differentiation secondary to these changes and thisprediction is confirmed.

Without wishing to be bound by theory, it is believed that materialsthat interfere specifically with regulation of sphingomyelin synthesisor degradation may prevent retrovirus-directed subrogation of immunesystem cellular function. The initial decrease in serum sphingomyelinlevel may be caused by (a) increased degradation or (b) decreasedsynthesis. The initial target for therapy would be determined by theactual mechanism.

One of the first biochemical actions of HIV during infection is known tobe decreased differentiation of CD4+ lymphocytes while later in thecourse of infection there is increased differentiation of CD8+lymphocytes. Both of these effects may be mediated by changes insphingomyelin concentration that are first reported in this application.Hence, enzymes controlling sphingomyelin levels present a new target totreat HIV infected individuals. Materials that bind to one of theenzymes and prevent the action of HIV on that enzyme would be ofparticular interest because they may prevent cell to cell transmissionand thus permit non-antibody based immune function to eliminate infectedcells. This type of process would lead to actual elimination of theinfection rather than just its suppression.

What is claimed is:
 1. A method for identifying a patient infected withHIV-1 comprising the steps of:detecting the amount of an agent selectedfrom the group consisting of sphingomyelin, sphingosyl-phospho-cholineand ceramides in a serum sample from said patient; wherein said patientis HIV-1 infected if the amount of said agent in said patient's serumsample is statistically different from the amount of said agent in serumsamples obtained from a control, non-HIV-1 infected group.
 2. A methodas defined in claim 1, wherein said statistical difference is aboutthree standard deviations.
 3. A method as defined in claim 1, whereinsaid agent detecting step is performed by antibody-based assay.
 4. Amethod as defined in claim 3, wherein said antibody-based assay isselected from the group consisting of IRMA, EIA, RIA and ELISA.
 5. Amethod as defined in claim 1, wherein said detecting step is performedwithin about one week of infection with HIV-1 in said patient.
 6. Amethod as defined in claim 5, wherein said detecting step is performedwithin about two days of infection with HIV-1 of said patient.
 7. Amethod for identifying a patient infected with HIV-1 comprising thesteps of:a. obtaining a serum sample from said patient; b. detecting theamount of an agent selected from the group consisting of sphingomyelin,sphingosyl-phospho-choline and ceramides in said sample; and c.comparing the amount of said agent in said sample with the amount ofsaid agent present in a serum sample obtained from an age-matchedcontrol group of non-HIV-1 infected individuals; wherein said patient isHIV-1 infected if the amount of said agent in said patient's sample isstatistically different from the amount of said agent in said controlgroup.
 8. A method for determining the efficacy of the treatment of ahuman patient infected with HIV-1 comprising the steps of:a. detectingthe amount of an agent selected from the group consisting ofsphingomyelin, sphingosyl-phospho-choline and ceramides in a serumsample from said patient before said treatment; and b.i. detecting theamount of said agent in a serum sample from said patient during saidtreatment; or ii. detecting the amount of said agent in a serum samplefrom said patient after said treatment; wherein said treatment iseffective if the amount of said agent in said sample of step a. isstatistically different from the amount of said agent in said sample ofstep b.
 9. A method as defined in claim 8, wherein said statisticaldifference is about three standard deviations.
 10. A method as definedin claim 8, wherein said agent quantifying steps a., b., or a. and b.are performed by antibody-based assay.
 11. A method as defined in claim10, wherein said antibody-based assay is selected from the groupconsisting of IRMA, EIA, RIA and ELISA.
 12. A method as defined in claim8 wherein said treatment comprises the administration of a memberselected from the group consisting of AZT, ddI, ddC or mixtures thereofto said patient.
 13. A method for determining the efficacy of thetreatment of a human patient infected with HIV-1 comprising the stepsof:a. obtaining a serum sample from said patient before said treatment;b. detecting the amount an agent selected from the group consisting ofsphingomyelin, sphingosyl-phospho-choline and ceramides in said sampleof step a.; c.i. obtaining a serum sample from said patient during saidtreatment; or ii. obtaining a serum sample from said patient after saidtreatment; and d. detecting the amount of said agent in said sample ofstep c.; e. comparing the amount of said agent in said sample of step a.with the amount of said agent in said sample of step c; wherein saidtreatment is effective if the amount of said agent in said sample ofstep a. is statistically different from the amount of said agent in saidsample of step c.
 14. A method for identifying a patient infected withHIV-1 comprising the steps of:obtaining a serum sample from saidpatient, detecting the amount of an agent selected from the groupconsisting of sphingomyelin, sphingosyl-phospho-choline and ceramides insaid serum sample, comparing the amount of said agent in said serumsample with that of the amount of said agent present in a serum sampleobtained from age-matched control, non-HIV-1 infected group, whereinsaid patient is HIV-1 infected if the amount of said agent in saidpatient's serum sample is statistically different from the amount ofsaid agent in said control, non-HIV infected group.
 15. The method ofclaim 14 wherein said statistical difference is about three standarddeviations.
 16. The method of claim 14 wherein said agent quantifyingstep is performed by antibody-based assay.
 17. The method of claim 14wherein said agent is quantified using an antibody-based test selectedfrom this group consisting of IRMA, EIA, RIA and ELISA.